There are 3 major objectives of this project, centered about the biochemical basis of respiratory energy transduction by the mitochondria of animal tissues. The first is the determination of the number, site, and mechanism by which H+ and electric charges are translocated across the inner mitochondrial membrane as each pair of electrons passes from organic substrate to O2 via the electron transport chain. Special attention will be placed on the vectorial processes associated with the cytochrome oxidase reaction and how it responds to intracellular concentrations of ATP, ADP, and phosphate, as well as 02. The second objective is to determine the mechanism and modes of regulation of inward and outward transport of essential metabolites by respiring mitochondria, particularly at intracelluar conditions. Special attention will be given to the respiration-dependent transport of Ca++ at the very low Ca++ concentrations in intact cells, as well as its biological regulation. Also to be examined is the transport of CO2 and bicarbonate and its effect on mitochondrial respiration and bioenergetics. Our third objective is to determine the mode of biosynthesis of 3-phosphocitrate, a mitochondrial inhibitor of calcium phosphate crystalization, which we have found inhibits kidney calcification in vivo. Its concentration and mode of action in cells and mitochondria will be investigated.